Project Summary Alcohol abuse is a major risk factor in initiating pancreatitis in humans and in rodents. Experimental evidence indicates that alcohol consumption leads to the destabilization of lysosomes and increases zymogen fragility in acinar cells. Cells can adapt and protect themselves by removing these damaged lysosomes and zymogen granules (ZGs) by autophagy and lysosomal biogenesis. However, these protective mechanisms are impaired after chronic alcohol consumption. Cannabinoid receptor 2 (CB2) agonists have potent anti-inflammatory effects and protect against experimental pancreatitis in mice. However, the underlying molecular mechanisms by which CB2 agonists protect against pancreatitis are not known. Moreover, what is also missing is the availability of CB2 agonists with optimal pharmacokinetics and absence of major toxicity as tested in relevant alcoholic pancreatitis models. In our preliminary studies, we established a novel alcohol pancreatitis mouse model using the recently established chronic plus acute alcohol binge (Gao-binge) protocol. Gao-binge induces significant pathogenesis in mouse pancreas, including edema, increased ZGs, elevated expression of inflammatory cytokines, increased infiltration of inflammatory cells and release of pancreatic enzymes (amylase and lipase) into the blood. We also demonstrated that Gao-binge decreases expression of pancreatic cannabinoid receptor 2 (CB2) and inactivates TFEB, a master regulator of lysosomal biogenesis. A selective CB2 agonist activates TFEB in mouse pancreas and increases autophagic flux in cultured acinar cells. Here we propose a novel hypothesis that ethanol metabolism leads to decreased CB2 expression and impaired TFEB-mediated lysosomal biogenesis, insufficient autophagy, accumulation of fragile ZGs, damaged lysosomes, and subsequent pancreatitis. Our long-term goal is to understand the molecular mechanisms for alcohol impairment of lysosomal biogenesis in pancreatic acinar cells, in order to identify steps in the repair pathway that are points for intervention in alcoholic pancreatitis. The objective of this proposal is to understand how CB2 agonists activate TFEB-mediated lysosomal biogenesis and selective zymophagy and lysophagy to protect against alcohol-induced pancreatitis. Using complimentary expertise in CB2 receptor pharmacology/toxicology (NIAAA intramural research program) and modeling of lysosomal biogenesis and alcoholic pancreatitis in vitro and in vivo (University of Kansas Medical Center) we propose to test a set of novel, highly selective and orally available CB2 agonists assuring quick translation toward clinical application for the treatment of alcoholic pancreatitis. We will determine how the selective CB2 agonists activate TFEB- mediated lysosomal biogenesis and their ability to improve the insufficient autophagy to remove damaged lysosomes and ZGs induced by chronic alcohol consumption. This proposal brings together research expertise that will address key research questions on alcoholic pancreatitis and the potential intervention by CB2 agonists, which would not otherwise be possible.